Gene therapy is likely to become the most significant development in medicine of our time. However, before gene therapy becomes a standard medical procedure, certain technical problems common to all methods of gene delivery must be overcome. One key obstacle is the current inability to produce large quantities of pure replication defective viral vectors.
Indeed, most gene therapy protocols use replication defective viral vectors as gene therapy vehicles. This is due to the ability of viruses to efficiently transfect their own DNA into a host cell. By replacing viral genes that are needed for the replication (the non-essential genes) with heterologous genes of interest, replication defective viral vectors can transduce the host cell and thereby provide the desired genetic material to the host cell. The non-essential genes can be provided in trans in order to produce the replication defective viral vectors. Thus the non-essential genes are placed into the genome of the packaging cell line, on a plasmid, or a helper virus. A number of replication defective viral vectors have been constructed, though most of the work has centered on three particular DNA viruses; the adenovirus, the adeno-associated virus and the herpes simplex virus type 1.
Adeno-associated virus type 2 (AAV) is a human non-pathogenic parvovirus with a genome of approximately 4.7 kb. The AAV genome consists of two ORFs that encode regulatory (Rep) and structural capsid (Cap) proteins flanked by 145-bp inverted terminal repeats (ITR). These ITRs are the only cis-acting elements necessary for virus replication and encapsidation. Recombinant AAVs (rAAV) which do not contain any endogenous coding regions efficiently propagate when Rep and Cap are provided in trans. In nature, a secondary infection with helper virus, e.g. adenovirus, is necessary to trigger a productive infection. AAV genomes then undergo replication followed by assembly of infectious virions containing ssDNA of either (+) or (−) polarity. Adenovirus genes implicated in AAV replication have been identified and include E1A, E1B, E4orf6, E2A, and VA RNA.
Similar to provirus in latently infected cells, AAV genomes can be efficiently rescued from a recombinant cis-plasmid following transient transfection into human cells. The necessary helper functions can be delivered either by adenovirus infection or by transfecting a plasmid encoding a minimal set of adenovirus helper genes (Collaco et al. (1999) Gene 238:397-405).
Events of AAV lytic infection are described by a commonly accepted self-priming strand-displacement model. The first 125 nucleotides of AAV termini include elements capable of forming a T-shaped duplex structure (A′-B′-B-C′-C-A) and are followed by a unique 20 bp D-sequence (Wang et al. (1995) J. Mol. Biol. 250:573-580). The Rep gene encodes four proteins that are synthesized from the same ORF via the use of alternate promoters and splicing. Two of these proteins (Rep78 and Rep68) possess site-specific and strand-specific endonuclease activity. They bind to the Rep-binding site (Rbs) mapped to the tetrameric GAGC repeat of the A-stem of the ITR and cleave it at the terminal resolution site (trs), positioned between the A- and D-elements. A tip of the BB′ palindrome contains RBE′, a cis-acting element essential for optimal Rep-specific activity. During replication, the terminus folds on itself and serves as a primer to initiate a leading-strand synthesis. At the elongation step, the complementary strand is displaced and may serve as an independent second replication template. The result of this first round of DNA synthesis is a linear duplex replication form monomer (Rfm) with a covalently closed hairpin on one end. Rep-mediated nicking of the original strand then creates a 3′-OH primer and the hairpin is extended. If nicking and subsequent ITR repair do not occur before the second round of replication is initiated on an opposite newly formed 3′ end, then continued DNA synthesis leads to formation of a replication form dimer (Rfd), which can be organized head-to-head (H-H) or tail-to-tail (T-T), but never head-to-tail. The model also predicts that linear duplex structures are intermediates of packaging. Using these as a template, the other two Rep proteins (Rep52 and Rep40) generate single-stranded progeny genomes which are then encapsidated into preformed capsids.
One of the great challenges in effectively applying gene therapy to human disease is the development of simple systems for rapidly generating high volumes of high titer viruses completely uncontaminated by potentially toxic helper viruses. One approach has been the development of techniques for producing “defective” viral vectors devoid of helper viruses. The most popular vectors include adeno-associated virus (AAV) and “gutless” adenovirus vectors which contain only the ITRs and a packaging sequence round the transgene. These harbor no viral genes, are incapable of replication, and helper viruses can be completely eliminated. Current strategies for producing such vectors, however, rely on techniques which either limit viral titers or which are so labor and resource intensive that they severely limit the clinical and commercial viability of these promising systems.
In an attempt to overcome this critical problem, new approaches have been attempted, though heretofore with limited success. In one such approach, a herpes amplicon system was created in which essential AAV genes (Rep and Cap) were inserted into the amplicon, and a second “helper” herpes simplex virus (HSV) was used to package the amplicon. The mix was then used to package AAV vector. This helper HSV virion was a mutant HSV that contained a mutation in a gene that is necessary for HSV infection, i.e., the glycoprotein H (gH) (Zhang et al., 1999, Hum. Gen. Ther. 10(15):2527-2537).
U.S. Pat. No. 5,139,941 (Muzyczka et al.) describes a AAV vector having the first and last 145 bp containing the ITRs, and capable of tranducing foreign DNA into a mammalian cell. U.S. Pat. No. 5,478,745 (Samulski et al.) describes a 165 bp fragment containing an AAV 145 bp ITR sequence with the 20 bp D sequence found to provide sufficient information in cis for replication and encapsidation of recombinant DNA fragments into mature AAV virions. U.S. Pat. No. 5,436,146 (Shenk et al.) describe helper free stocks of recombinant AAV vectors. Collaco et al. (1999) Gene 238:397-405 describe a helper virus-free packaging system for recombinant AAV vectors.